Radioimmunological quantitation of urinary trypsin inhibitor

Using monospecific antibody to human urinary trypsin inhibitor, we developed a highly specific and sensitive radioimmunoassay (RIA) for measuring human urinary trypsin inhibitor. No cross-reactivity of the antibody with protein standard serum, which contained albumin, alpha 1-antitrypsin, haptoglobin, alpha 2-macroglobulin, transferrin, IgG and IgA, was observed. The sensitivity of the system was 10 ng of trypsin inhibitor per assay tube, and 5-10 microliters of urine was sufficient to determine the concentration of trypsin inhibitor in urine. The amounts excreted in the urine of 10 healthy men and 10 healthy women were 4.83 +/- 2.46 (mean +/- SD) and 3.86 +/- 1.35 mg/day, respectively. The correlation between estimates by RIA and those by enzymic assay was r = 0.96 (p less than 0.005). The method proposed here can be used to determine the concentration of urinary trypsin inhibitor in a small amount of biological fluids and cells.     

Immunochemical studies of human urinary trypsin inhibitor

Antisera against purified urinary trypsin inhibitor (UTI-I, molecular weight 67,000) and UTI-III (molecular weight 23,000) were first produced in rabbits. Both anti-UTI-I and anti-UTI-III sera formed a single immunoprecipitin line with human plasma inter-alpha-trypsin inhibitor (I alpha TI), whereas two immunoprecipitin lines were formed with crude urine. It was speculated that both UTI-I and UTI-II might be present in normal human urine. In the present study, the inhibitory effects of anti-UTI sera on UTI activity were examined by three different assay methods. The results indicated that the inhibitory effect was almost immediate. Although the inhibitory effect of anti-UTI-III serum on UTI-III was almost of the same degree of completeness for the three assay methods. UTI-I was partially inhibited by the anti-UTI-I serum when residual trypsin activity was measured by the caseinolytic or fibrinolytic assay method. This discrepancy was considered to be due to the difference in conformational change between UTI-I and UTI-III by antigen-antibody reaction.     

Human granulocyte elastase is inhibited by the urinary trypsin inhibitor

Two forms of urinary trypsin inhibitor, A and B, were purified from the urine of pregnant women. Form A was the only inhibitor present in fresh urine and inhibitor B arose from degradation of A upon storage of urine. The molecular masses of A and B were about 44 and 20 kDa, respectively, as judged from dodecyl-sulfate polyacrylamide gel electrophoresis, but about 60 kDa and 30 kDa, respectively, as judged from gel filtration analysis. The discrepancy can perhaps be explained by the carbohydrate content amounting to about 10% of each inhibitor. After reduction with mercaptoethanol, inhibitor A and inhibitor B had identical apparent molecular masses of about 20 kDa on dodecyl-sulfate gel electrophoresis. These results and the results of amino acid analysis suggest that one molecule of inhibitor A yields two molecules of inhibitor B. On agarose gel electrophoresis inhibitor A migrated as a rather broad band in the prealbumin region and inhibitor B as 3 well defined bands in the beta-region. Specific antisera were raised against inhibitor A and B. The two inhibitors showed the immunologic reaction of identity with each other and with the plasma inter-alpha-trypsin inhibitor, when using either antiserum. The inhibitors both gave quantitative inhibition of bovine trypsin, the results indicating a 4/1 trypsin/inhibitor molar ratio for A and a 2/1 ratio for B. The two substances also effectively inhibited granulocyte elastase. No inhibition of porcine pancreatic elastase was demonstrable.     

The major human urinary trypsin inhibitor is a proteoglycan

The major urinary trypsin inhibitor (Mr 44 000), isolated from human urine, contains 35% carbohydrate. In addition to N-acetylglucosamine and neutral sugars (primarily mannose and galactose), the carbohydrate moiety contains hexuronic acid and N-acetylgalactosamine and corresponds to a glycosaminoglycan. This carbohydrate chain is an integral component of the inhibitor: it does not dissociate from the inhibitor when using dissociative conditions such as sodium dodecyl sulfate, guanidinium chloride, or by increasing ionic strength or mixing with cetylpyridinium chloride. This glycosaminoglycan chain is sensitive to chondroitinase ABC or testicular hyaluronidase digestion and corresponds to slightly sulfated chondroitin 4-sulfate or 6-sulfate. After treatment by these enzymes, the urinary inhibitor has a lower molecular mass (Mr 26 000) but still inhibits trypsin.     

Impaired fertility in female mice lacking urinary trypsin inhibitor

Urinary trypsin inhibitor (UTI) is a serine proteinase inhibitor that is found in blood and urine. To investigate the physiological functions of UTI in vivo, we generated UTI-deficient mice by gene targeting. The mice showed no obvious abnormalities and appeared healthy. However, the females displayed a severe reduction in fertility. Wild-type embryos developed normally when transplanted into UTI-deficient female mice, suggesting that UTI-deficient females have a normal ability to maintain pregnancy. The number of naturally ovulated oocytes from UTI-deficient mice was greatly reduced compared with that from wild-type mice. Histologically, oocytes with disorganized corona radiata were frequently seen in the ovaries of UTI-deficient mice after hormonal stimulation. When ovaries from UTI-deficient mice were transplanted into wild-type mice, pups derived from the transplanted ovaries were obtained, suggesting that the ovary of UTI-deficient mice functions normally if UTI is supplied from the systemic circulation. These results demonstrate that UTI plays an important role in the formation of the stable cumulus-oocyte complex that is essential for oocyte maturation and ovulation.     

Multiple molecular forms of acid-stable plasmin inhibitor derived from human urinary trypsin inhibitor

It was found that cyanogen bromide (BrCN) treatment of the highly purified human urinary trypsin inhibitors (H-UTI; specific activity 1,897 U/mg protein, and L-UTI; specific activity 1,850 U/mg protein) readily produced new plasmin inhibitors with almost no loss of UTI activity. Five multiple forms of chemically cleaved inhibitors (UTIB-I, UTIB-II, UTIB-III, UTIB-IV and UTIB-V) could be isolated from BrCN-treated L-UTI by isoelectric focusing and gel filtration. These inhibitors were very acid-stable and their isoelectric points (pI) were 4.5, 4.6, 4.9, 5.1 and 6.4, respectively. The molecular weights by SDS-polyacrylamide gel electrophoresis were almost the same at about 23,000 +/- 3,000. Although these inhibitors showed both anti-plasmin and anti-trypsin activities, much higher anti-plasmin/anti-trypsin activities were observed in the cleaved inhibitors than in the parent UTI. They competitively inhibited human plasmin with Ki values of 3.0-4.1 X 10(-8) mol/l (H-D-Val-Leu-Lys-pNA substrate).     

Calcium-induced changes in chondroitin sulfate chains of urinary trypsin inhibitor

Urinary trypsin inhibitor (UTI) has several roles other than protease inhibition. It is suggested that UTI inhibits calcium influx in cultured cells and that the chondroitin sulfate chain of UTI may play an important role. In order to clarify the mechanistic features of this phenomenon, the chondroitin sulfate chain of UTI was analyzed by (1)H-NMR. The samples were highly purified UTI dissolved in D(2)O in the presence or absence of Ca(2+), Mg(2+) and Na(+). 1D-spectra were obtained and T(1) values of detected signals were estimated from the inversion-recovery method. The addition of Ca(2+) to UTI caused a chemical shift to downfield, line broadening and a reduction of T(1) values at several signals from chondroitin sulfate moiety (especially at axial H-2 of GalNAc), whereas Mg(2+) and Na(+) had no significant effect. Some of the signals in the linkage region of chondroitin sulfate chain showed marked line broadening by Ca(2+). The reduction of T(1) values implies formation of a complex. It is suggested that Ca(2+) generates the sulfate salt and interacts with other polar groups in the chondroitin sulfate chain, thereby causing bridging between UTI molecules. Several properties of UTI may be related to this interaction of Ca(2+) with chondroitin sulfate chains.     

Radioimmunological quantitation of the urinary trypsin inhibitor in normal blood and urine

A radioimmunoassay for measurement of the urinary trypsin inhibitor in human serum and urine is described. Because of the immunological cross-reactivity between the inter-alpha-trypsin inhibitor and the urinary trypsin inhibitor the plasma and serum were treated with perchloric acid to precipitate the inter-alpha-trypsin inhibitor. Gel filtration of serum before and after acid treatment showed identical peaks corresponding to the urinary trypsin inhibitor. The normal level of the urinary trypsin inhibitor in fresh plasma from 30 blood donors was 6.38 +/- 0.33 mg/l (SEM), and in sera from 24 healthy volunteers 7.14 +/- 0.27 mg/l (SEM). In urine from 23 healthy volunteers the normal excretion was 8.17 +/- 1.18 mg/24 h (SEM).     

Purification and partial characterization of two forms of urinary trypsin inhibitor

The activation of bovine thyroid adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by Gpp(NH)p has been studied using steady-state kinetic methods. This activation is complex and may be characterized by two Gpp(NH)p binding sites of different affinities with measured constants: Ka1 = 0.1 micro M and Ka2 = 2.9 micro M. GDP beta S does not completely inhibit the Gpp(NH)p activation: analysis of the data is consistent with a single GDP beta S inhibitory site which is competitive with the weaker Gpp(NH)p site. Guanine nucleotide effects upon F- activation of adenylate cyclase have been studied. When App(NH)p is the substrate, 10 micro M GTP along with 10 mM NaF gives higher activity than NaF alone, while GDP together with NaF inhibits the activity by 50% relative to NaF. These features are not observed when the complex is assayed with ATP in the presence of a nucleotide regenerating system or when analogs Gpp)NH)p or GDP beta S are used along with NaF. These effects were studied in three other membrane systems using App(NH)p as substrate: rat liver, rat ovary and turkey erythrocyte. No consistent pattern of guanine nucleotide effects upon fluoride activation could be observed in the different membrane preparations. Previous experiments showed that the size of soluble thyroid adenylate cyclase changed whether membranes were preincubated with Gpp(NH)p or NaF. This size change roughly corresponded to the molecular weight of the nucleotide regulatory protein. This finding, coupled with the present data, suggests that two guanine nucleotide binding sites may be involved in regulating thyroid cyclase and that these sites may be on different protein chains.     

On the carbohydrate composition of bovine colostrum trypsin inhibitor.

The trypsin inhibitor from bovine colostrum has been separated into several forms by CM-Sephadex and DEAE-cellulose chromatography. These forms differ in the amount and composition of the carbohydrate they contain, which has been quantitated for four components by gas-liquid chromatography and standrad colorimetric procedures. The monosaccharides fucose, mannose, galactose, galactosamine, glucosamine and sialic acids have been determined. A microheterogeneity was establish ed in the carbohydrate moiety, which amounts to about 40% of the total molecular weight (Mr 11 000 - 14 000) of bovine colostrum inhibitor.     

Cytoprotective effects of urinary trypsin inhibitor on astrocytes injured by sustained compression

Decreased cell membrane integrity is a primary pathological change observed in traumatic brain injury (TBI) that activates a number of complex intercellular and intracellular pathological events, leading to further neural injury. In this paper, we assessed the effects of urinary trypsin inhibitor (UTI) on astrocyte membrane integrity by determining the percentage of lactate dehydrogenase (LDH) released after sustained compression injury using a hydrostatic pressure model of mechanical-like TBI. Astrocytes isolated from SD rat pups were injured by sustained compression. At a pressure of 0.3 MPa for 5 min, a significant increase in LDH release was observed compared with control samples. Astrocytes displayed extensive structural disruption of mitochondrial cristae reflected in their swelling. Based on our initial results, injured astrocytes were treated with UTI at a final concentration of 500, 1,000, 3,000 or 5,000 U/ml for 24 h. The percentage of LDH released from injured astrocytes was significantly decreased when 1,000 and 3,000 U/ml of UTI were used. In a separate experiment, astrocytes were treated with UTI at a final concentration of 1,000 U/ml immediately, or at 30 min, 2, 6, or 24 h after sustained compression. The percentage of LDH release was significantly reduced (P < 0.05) when astrocytes were treated with UTI immediately or 30 min later. Together, our results suggest that UTI may have protective effects on astrocytes injured by sustained compression injury. Furthermore, the early administration (<2 h after injury) of UTI may result in a better outcome compared with delayed administration.     

Biosynthesis of bikunin (urinary trypsin inhibitor) in rat hepatocytes.

One of the major sulfated proteins secreted by rat hepatocytes contains a low-sulfated chondroitin sulfate chain and its apparent molecular mass upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis shifts from 40 to 28 kDa upon chondroitinase ABC treatment (E. M. Sj?berg and E. Fries, 1990, Biochem. J. 272, 113-118). These properties suggest that this protein is the rat homologue of the major trypsin inhibitor of human urine which was recently named bikunin. In serum, bikunin occurs mainly as a subunit of the pre-alpha-inhibitor and the inter-alpha-inhibitor; in these proteins it is covalently linked to the other polypeptides through its chondroitin sulfate chain. Bikunin has been shown to be synthesized by liver cells as a 42-kDa precursor, in which it is linked to alpha 1-microglobulin by two basic amino acids. We have isolated bikunin from rat urine and prepared antibodies against it. In rat hepatocytes pulse-labeled with [35S]methionine, these antibodies precipitated a labeled protein of 42 kDa. Upon chase, three different labeled proteins were recognized by the antibodies in the medium: one protein of 40 kDa (free bikunin), one of 125 kDa (presumably pre-alpha-inhibitor), and one greater than 240 kDa (possibly a protein related to the inter-alpha-inhibitor). Pulse-chase experiments with [35S]sulfate showed that these proteins occurred intracellularly as precursors containing alpha 1-microglobulin. These results demonstrate that the completion of the chondroitin sulfate chain and its coupling to other polypeptide chains occur before the cleavage of the alpha 1-microglobulin/bikunin precursor.     

A chondroitin-sulfate chain is located on serine-10 of the urinary trypsin inhibitor.

1. The glycopeptide carrying the glycosaminoglycan chain of the urinary trypsin inhibitor (immunologically and structurally related to inter-alpha-trypsin inhibitor) was isolated. 2. The data from amino acid composition and part sequencing of this glycopeptide unambiguously demonstrate that the glycosaminoglycan is covalently linked to serine-10 of the peptide chain of UTI.     

A proteoglycan related to the urinary trypsin inhibitor (UTI) links the two heavy chains of inter-alpha-trypsin inhibitor

cDNA studies have suggested that inter-alpha-trypsin inhibitor (ITI) is a complex of several different peptide chains; the sequence of the inhibitory part of ITI is in excellent agreement with that of the urinary trypsin inhibitor (UTI). The present report demonstrates that a compound immunologically related to UTI is released by digestion with porcine pancreatic elastase or human leucocyte elastase. Since UTI has been shown to be a proteoglycan, ITI has been treated by chondroitinase. In these conditions, ITI is dissociated and gives rise to two heavy chains (78 and 85 kDa) and one light chain (26 kDa) immunologically related to UTI and which in PAGE moves close to UTIc (produced by chondroitinase treatment of UTI). We suggest that ITI is a non-covalent complex comprising two heavy chains and one light chain immunologically related to UTI and which is also a proteoglycan.     

Hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor beta-sheet protons in trypsin-bovine pancreatic trypsin inhibitor, trypsinogen-bovine pancreatic trypsin inhibitor, and trypsinogen-isoleucylvaline-bovine pancreatic trypsin inhibitor

Hydrogen exchange rates of six beta-sheet peptide amide protons in bovine pancreatic trypsin inhibitor (BPTI) have been measured in free BPTI and in the complexes trypsinogen-BPTI, trypsinogen-Ile-Val-BPTI, bovine trypsin-BPTI, and porcine trypsin-BPTI. Exchange rates in the complexes are slower for Ile-18, Arg-20, Gln-31, Phe-33, Tyr-35, and Phe-45 NH, but the magnitude of the effect is highly variable. The ratio of the exchange rate constant in free BPTI to the exchange rate constant in the complex, k/kcpIx, ranges from 3 to much greater than 10(3). Gln-31, Phe-45, and Phe-33 NH exchange rate constants are the same in each of the complexes. For Ile-18 and Tyr-35, k/kcpIx is much greater than 10(3) for the trypsin complexes but is in the range 14-43 for the trypsinogen complexes. Only the Arg-20 NH exchange rate shows significant differences between trypsinogen-BPTI and trypsinogen-Ile-Val-BPTI and between porcine and bovine trypsin-BPTI.     

Human urinary proteinase inhibitor: inhibitory properties and interaction with bovine trypsin

The major urinary trypsin inhibitor (UTI) was found to inhibit bovine chymotrypsin and human leucocyte elastase strongly, cathepsin G weakly. No inhibition of porcine pancreatic elastase was observed. The stoichiometry of the inhibition of bovine trypsin by UTI was determined spectrophotometrically to be 1:2 (I/E molar ratio). After incubation of UTI with this enzyme in various molar ratios, two complexes (C1 and C2) could be visualized in alkaline polyacrylamide gel electrophoresis. C1 was isolated by affinity chromatography on Con-A Sepharose. In dodecyl sulfate polyacrylamide gel electrophoresis, C1 was dissociated to give an inhibitory band with the same electrophoretic mobility as native UTI. C2 released an active inhibitory fragment with Mr near 20000. A time-course study demonstrated that at a molar ratio I/E of 1.5:1, the C2 complex appears after two hours of incubation.     

Protective role of urinary trypsin inhibitor in acute lung injury induced by lipopolysaccharide

Urinary trypsin inhibitor (UTI), a serine protease inhibitor, has been widely used as a drug for patients with acute inflammatory disorders such as disseminated intravascular coagulation, shock, and pancreatitis. However, direct contribution of UTI to inflammatory diseases has not been established. The present study analyzed acute inflammatory lung injury induced by lipopolysaccharide (LPS) in UTI-deficient (-/-) mice and corresponding wild-type (WT) mice. UTI (-/-) and WT mice were treated intratracheally with vehicle or LPS (125 mug/kg). The cellular profile of bronchoalveolar lavage fluid, lung water content, histology, and expression of proinflammatory molecules in the lung were evaluated. After LPS challenge, both genotypes of mice revealed neutrophilic lung inflammation and pulmonary edema. UTI (-/-) mice, however, showed more prominent infiltration of inflammatory cells and edema than WT mice. After LPS challenge in both genotypes of mice, the lung levels of mRNA and/or protein expression of interleukin-1beta, macrophage inflammatory protein-1alpha, macrophage chemoattractant protein-1, keratinocyte chemoattractant, and intercellular adhesion molecule-1 (ICAM-1) were elevated in both groups, but to a greater extent in UTI (-/-) mice than in WT mice. These results suggest that UTI protects against acute lung injury induced by bacterial endotoxin, at least partly, through the inhibition of the enhanced local expression of proinflammatory cytokines, chemokines, and ICAM-1.